Computational Design of Unnatural Amino Acid Dependent Metalloproteins

描述（由申请人提供）：非天然氨基酸的位点特异性掺入和计算蛋白质设计领域的融合代表了目前尚未探索但有前途的生物化学研究途径。虽然已经开发了用于天然存在的蛋白质的计算方法，但是用这些技术处理非天然氨基酸的能力尚未被充分探索。这项研究旨在开发一种计算方法，允许设计含有非天然氨基酸的蛋白质，最终目标是产生具有治疗潜力的新型非天然氨基酸依赖性酶。 由华盛顿大学贝克实验室成员开发的罗塞塔软件套件将首先用于设计利用金属结合非天然氨基酸联吡啶丙氨酸的铁结合蛋白-舒尔茨和斯克里普斯研究所的同事首次将其纳入蛋白质中。由于这种非天然氨基酸对铁具有固有的亲和力，因此设计蛋白质内的金属结合位点的难题应该在计算上更加易于处理。作为第二个目标，多巴胺的结合位点（将为铁提供两个氧配体）将同时工程化。像多巴胺这样的儿茶酚与铁有着天生的高亲和力，这表明这种工程蛋白质可以作为这类重要小分子的传感器。最后，儿茶酚结合蛋白将被进一步计算设计，目标是产生非天然氨基酸依赖性的二醇外双加氧酶样酶。这种酶可能对多氯联苯化合物等持久性拟人毒素的生物修复产生深远影响。 设计的含有非天然氨基酸的蛋白质将使用舒尔茨实验室成员开发的技术在细菌表达系统中产生。然后将使用大量生物分析技术分析纯化的蛋白质，这些技术将检查金属或儿茶酚结合能力或酶活性，这取决于具体目的。在实验过程中收集的数据将用于未来设计其他非天然氨基酸蛋白质，无论是在本项目的范围内，还是超越。 因此，这项研究应该在生物科学领域产生深远的影响，超出上述项目的范围。由于该提案中探索的两个科学领域目前都处于快速增长的状态，因此在本研究过程中收集的任何信息都将指导进一步的计算设计工作，涉及其他目前可用的遗传编码的非天然氨基酸以及未来开发的氨基酸。 公共卫生相关性：这项研究最终旨在工程化含有非天然氨基酸的蛋白质，这些蛋白质具有催化降解多氯联苯环境毒素的能力。此外，在研究过程中开发的计算方法将提供重要的信息，指导未来设计具有治疗和其他有用功能的含非天然氨基酸的蛋白质的工作。

DESCRIPTION (provided by applicant): The confluence of the fields of site-specific incorporation of unnatural amino acids and computational protein design represents a currently unexplored but promising avenue of biochemical research. While computational methods have been developed for naturally occurring proteins, the ability to treat non-natural amino acids with these techniques has yet to be fully explored. The research proposed seeks to develop a computational method that allows design of proteins containing unnatural amino acids, with the ultimate goal of generating novel unnatural amino acid dependent enzymes with therapeutic potential. The Rosetta suite of software developed by members of the Baker lab at the University of Washington will first be used to design iron binding proteins that utilize the metal binding unnatural amino acid bipyridyl alanine - first incorporated into proteins by Schultz and co-workers at The Scripps Research Institute. As this unnatural amino acid has inherent affinity for iron, the difficult problem of designing a metal binding site within a protein should be rendered more computationally tractable. As a second goal, a binding site for dopamine (which will provide two oxygen ligands for the iron) will be concurrently engineered. Catechols like dopamine have inherently high affinities for iron suggesting the engineered proteins could serve as sensors for this important class of small molecules. Finally, the catechol binding proteins will be further designed computationally with the goal of creating a non-natural amino acid dependent extradiol dioxygenase like enzyme. Such an enzyme could have a far-reaching impact with respect to bioremediation of persistent anthropomorphic toxins such as polychlorinated biphenyl compounds. The designed unnatural amino acid containing proteins will be produced in a bacterial expression system using techniques developed by members of the Schultz laboratory. Purified proteins will then be analyzed using a host of bioanalytical techniques that will examine metal or catechol binding abilities, or enzymatic activity depending on the specific aim. Data collected in the course of experimentation will be used for future design of other unnatural amino acid containing proteins both within the scope of this project, and beyond. Consequently, this research should have far reaching impacts within the biological sciences that will extend beyond the projects described above. As both of the scientific fields explored in this proposal are currently in a state of rapid growth, any information gleaned in the course of this research will guide further computational design efforts involving other currently available, genetically encoded non-natural amino acids, as well as those developed in the future. PUBLIC HEALTH RELEVANCE: This research ultimately seeks to engineer unnatural amino acid containing proteins that possess the ability to catalytically degrade polychlorinated biphenyl environmental toxins. Additionally, the computational methods developed in the course of the research will provide vital information that will guide future efforts for the design of unnatural amino acid containing proteins with therapeutic and other useful functions.